Understanding biogeographic structure is of fundamental importance for preserving biodiversity. However, large-scale phylogeographic information is difficult to obtain because it requires thousands of individual samples from large geographic areas. Since recent advances in environmental DNA (eDNA) analysis have enabled us to identify intra-specific genetic variations in aquatic organisms, it can also be applied to estimate phylogeographic patterns of freshwater fish without collecting individual fish samples. Here we show that an eDNA detection system we developed enabled us to investigate the phylogeography of a primary freshwater fish Barbatula toni in Hokkaido. After screening 297 river water samples throughout Hokkaido, we detected 50 haplotypes from 52 river systems. They were composed of six genetic groups with tight correspondence to their distributed regions. Notably, they were divided into two distinct genetic lineages without obvious geographic barriers, which was unique compared to other aquatic species inhabiting in Hokkaido. Together with the results of several analyses, we discuss the potential scenarios of population expansion and vicariance of B. toni. Our findings demonstrate the strong advantages of the eDNA technique as an innovative population genetic method to assess biodiversity at a large scale.